An anti-idiotypic antibody that neutralizes anti-D antibody and its applications

Abstract

The present invention discloses an anti-idiotypic antibody that neutralizes anti-D antibodies, which comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region contains HCDR1, HCDR2, and HCDR3 sequences, and the light chain variable region contains LCDR1, LCDR2, and LCDR3 sequences, wherein: the amino acid sequence of HCDR1 is as shown in SEQ ID No.1; the amino acid sequence of HCDR2 is as shown in SEQ ID No.2; the amino acid sequence of HCDR3 is as shown in SEQ ID No.3. The amino acid sequence of LCDR1 is as shown in SEQ ID No.4; the amino acid sequence of LCDR2 is as shown in SEQ ID No.5; the amino acid sequence of LCDR3 is as shown in SEQ ID No.6. This anti-idiotypic antibody can specifically bind to anti-D antibodies and can be applied to clinical blood group detection or the prevention or treatment of anti-D antibody-related diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to an anti-idiotypic antibody that neutralizes anti-D antibodies and its applications. Background Art

[0002] In clinical blood transfusion therapy, the Rh blood group system is second only to the ABO blood group system in importance. The RH alleles encode a total of 55 antigens. The polymorphism of the RH gene results in the polymorphism of Rh antigens, making it one of the most complex systems among the 47 human red blood cell blood group systems.Rh blood group system antigens have strong immunogenicity, among which the D antigen has the strongest immunogenicity. Some studies have found that Rh-negative individuals may produce anti-D when they receive more than 2 ml of Rh-positive red blood cells for the first time. In recent years, with the progress of experimental techniques and data sharing, it has been found that not only Rh-negative (D antigen-deficient) individuals can produce anti-D, but also some D variant individuals with incomplete expression of the D antigen due to RHD gene mutations can produce anti-D under the stimulation of the D antigen due to the deletion of D antigen epitopes. Among the population, the relatively common weak D15 type, RHD-CE(2-9)-D fusion type, DVI-Ⅲ type, etc., all have literature reports of producing anti-D. Anti-D is mainly an IgG-type immune antibody, which can be produced through immune pathways such as blood transfusion or pregnancy. Anti-D can cause acute or chronic hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (HDFN). Therefore, in clinical work, accurately detecting anti-D is the basic guarantee for blood transfusion safety and also the basis for guiding the prevention, diagnosis and treatment of Rh-HDFN. At present, the method for detecting IgG-type anti-D is the indirect antiglobulin test, which uses a secondary antibody against the Fc end as a mediator to cause agglutination of red blood cells bound with IgG antibodies. The gold standard for detecting antibodies is the tube indirect antiglobulin test, but it has the disadvantages of cumbersome operation and being greatly affected by the technical level of operators. The concentration of anti-D is usually expressed as titer or dilution. The detection method is to dilute the antibody and detect it by the indirect antiglobulin test, which cannot accurately measure the amount of anti-D and also has the disadvantage of rough results. Summary of the Invention

[0003] The purpose of the present invention is to provide an anti-idiotypic antibody that neutralizes anti-D antibodies and its applications. This anti-idiotypic antibody can specifically bind to anti-D antibodies and can be applied to clinical blood group detection or play a role in preventing or treating anti-D antibody-related diseases.

[0004] The present invention adopts the following technical scheme: An anti-idiotypic antibody that neutralizes anti-D antibodies, including a heavy chain variable region and a light chain variable region. The above heavy chain variable region contains at least one of the HCDR1, HCDR2, and HCDR3 sequences, wherein:

[0005] The amino acid sequence of the above HCDR1 has the amino acid sequence shown in SEQ ID No.1;

[0006] The amino acid sequence of the above HCDR2 has the amino acid sequence shown in SEQ ID No. 2;

[0007] The amino acid sequence of the above HCDR3 has the amino acid sequence shown in SEQ ID No. 3;

[0008] or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the above SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, or an amino acid sequence having more than 90% identity with the above SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3.

[0009] Furthermore, the light chain variable region includes any one of the LCDR1, LCDR2 and LCDR3 sequences, wherein:

[0010] The amino acid sequence of the above LCDR1 has the amino acid sequence shown in SEQ ID No. 4; or

[0011] The amino acid sequence of the above LCDR2 has the amino acid sequence shown in SEQ ID No. 5; or

[0012] The amino acid sequence of the above LCDR3 has the amino acid sequence shown in SEQ ID No. 6;

[0013] or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the above SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6, or an amino acid sequence having more than 90% identity with the above SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6.

[0014] Furthermore, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 7; the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 8.

[0015] Furthermore, the anti-idiotype antibody is a murine antibody.

[0016] Furthermore, the constant region of the heavy chain of the anti-idiotype antibody is the heavy chain constant region of murine IgM, IgG1, IgG2a, IgG2b or IgG3, and the κ or λ type light chain constant region.

[0017] Furthermore, the antigen-binding portion of the anti-idiotype antibody is selected from F(ab’)2 fragments.

[0018] The present invention also discloses the use of the above-mentioned anti-idiotypic antibody for detecting and neutralizing anti-D antibody, the use of the above anti-idiotypic antibody for detecting the content of anti-D antibody in serum; or the use for neutralizing anti-D antibody.

[0019] The present invention also discloses the use of the above-mentioned anti-idiotypic antibody for detecting and neutralizing anti-D antibody, its use in preparing drugs for anti-D antibody-related diseases.

[0020] The beneficial effects of the present invention are as follows: 1. An anti-idiotypic antibody is obtained against the antigenic determinant of the variable region, which can specifically bind to the Fab end of the anti-D antibody and play the role of simulating an antigen. 2. This anti-idiotypic antibody has high affinity for the anti-D antibody, can block the erythrocyte agglutination mediated by the anti-D antibody, can be used to detect various mixed samples of unexpected blood group antibodies containing anti-D, and can also be used for the risk assessment and clinical diagnosis and treatment of Rh-HDFN, and has important application value for improving the safety of clinical blood transfusion and disease treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a test chart for the preparation of human anti-D antibody and the preparation of immunogen by pepsin digestion; wherein: A is the SDS-PAGE electrophoresis chart before and after protein purification of the crude product of the anti-D antibody erythrocyte eluate; B is the SDS-PAGE electrophoresis chart of the products before and after pepsin digestion of the anti-D antibody.

[0022] Figure 2 It is a chart for measuring the antibody titer of the serum of mice immunized with anti-D-F(ab’)2 protein.

[0023] Figure 3 It is a chart for screening anti-idiotypic antibody by enzyme-labeling method.

[0024] Figure 4 It is an SDS-PAGE electrophoresis chart of anti-idiotypic antibody; wherein: M is the pre-stained protein Marker; 3 represents the 491# anti-idiotypic antibody.

[0025] Figure 5 It is a fitting chart of anti-idiotypic antibody detected by Fortebio.

[0026] Figure 6 It is a chart for the neutralizing effect of anti-idiotypic antibody on erythrocyte agglutination mediated by different anti-D antibodies; wherein: the anti-D antibodies selected are Shanghai anti-D antibody and Millipore anti-D antibody.

[0027] Figure 7 It is the identification of recombinant anti-idiotypic antibody; wherein A is the SDS-PAGE electrophoresis chart of anti-idiotypic antibody; B. The erythrocyte deagglutination experiment of recombinant anti-idiotypic antibody. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0029] In the anti-idiotypic antibody for neutralizing anti-D antibody and its application of the present invention, the term "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains, namely two heavy chains H and two light chains L interconnected by disulfide bonds, and its polymers, such as IgM. Each heavy chain comprises a heavy chain variable region and a heavy chain constant region, the heavy chain variable region is abbreviated as VH, and the heavy chain constant region is abbreviated as CH. The heavy chain constant region comprises three domains, namely CH1, CH2 and CH3. Each light chain comprises a light chain variable region and a light chain constant region, the light chain variable region is abbreviated as VL, and the light chain constant region is abbreviated as CL. The light chain constant region comprises one domain CL1. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions, the complementarity determining regions are written as CDR, and are interspersed with conserved regions called framework regions FR. The framework regions are written as FR. From the N-terminus to the C-terminus, both the light and heavy chain variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0030] The "antigen-binding portion" of an antibody refers to a part or segment of the intact antibody molecule responsible for binding to an antigen. The antigen-binding portion can comprise the heavy chain variable region, the light chain variable region, or both. The antigen-binding portion of an antibody can be prepared from the intact antibody molecule using any suitable standard techniques, including proteolytic digestion or recombinant genetic engineering techniques, etc. Non-limiting examples of antigen-binding portions include: Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-chain Fv (scFv) molecules, single-domain antibodies, dAb fragments, and minimal recognition units composed of amino acid residues mimicking the hypervariable regions of an antibody, such as isolated CDRs. The term "antigen-binding portion" also includes other engineered molecules, such as diabodies, triabodies, tetra-bodies, and minibodies, etc. For example, the Fd fragment in the present invention refers to an antibody fragment composed of the VH and CH1 domains; the Fv fragment is composed of the VL and VH domains in a single arm of an antibody; the dAb fragment is composed of the VH domain.

[0031] The complementarity determining region is the region in the variable region that has the greatest impact on the affinity and specificity of an antibody. CDRs usually include CDR1, CDR2 and CDR3; there are two common definitions for the CDR sequences of VH or VL, namely the Kabat definition and the Chothia definition. The CDR region sequences in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition. In the present invention, the Kabat definition is used to define the CDR sequences. In the present invention, the CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively abbreviated as HCDR1, HCDR2 and HCDR3; the CDR1, CDR2 and CDR3 of the light chain variable region are respectively abbreviated as LCDR1, LCDR2 and LCDR3.

[0032] For the variable region sequence of a given antibody, the CDR region sequences in the variable region sequence can be analyzed in various ways, for example, it can be determined by using the online software Abysis.

[0033] In the present invention, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigenic epitope, for example, the ability of an antibody to bind to a specific antigen with an affinity at least twice as great as its affinity for a non-specific antigen. However, it should be understood that an antibody can specifically bind two or more antigens related to its sequence. For example, the antibodies in the present invention can specifically bind human anti-D antibodies.

[0034] In the present invention, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, each antibody constituting the population is identical except for naturally occurring mutations that may be present in a small number of individuals. The monoclonal antibodies described herein specifically include "chimeric" antibodies, wherein a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and also includes fragments of such antibodies, provided that they can exhibit the desired biological activity.

[0035] In the present invention, the term "murine antibody" refers to any antibody in which all domain sequences are murine sequences. Such antibodies can be produced by hybridomas. The term "anti-D antibody-related diseases" includes diseases and / or disorders related to anti-D antibodies. Anti-D antibody-related diseases or disorders include one or more of hemolytic disease of the fetus and newborn.

[0036] In the present invention, the term "treatment" refers to any type of intervention or method performed on a subject or the administration of an active agent to it, wherein the purpose is to reverse, alleviate, improve, inhibit or relieve or prevent symptoms, complications, conditions or the progression, development, severity or recurrence associated with a disease. The term "prevention" refers to the administration to a subject not suffering from a disease to prevent the occurrence of the disease or, if present, to minimize its effects.

[0037] The present invention discloses an anti-idiotypic antibody that neutralizes anti-D antibodies, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises at least one of the HCDR1, HCDR2 and HCDR3 sequences, wherein:

[0038] The amino acid sequence of the HCDR1 has the amino acid sequence shown in SEQ ID No.1: DYTFTNYW.

[0039] The amino acid sequence of the HCDR2 has the amino acid sequence shown in SEQ ID No.2: IYLGDGDT.

[0040] The amino acid sequence of the HCDR3 has the amino acid sequence shown in SEQ ID No. 3: ARDGWYFDV.

[0041] Or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, or an amino acid sequence having more than 90% identity with SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3.

[0042] The light chain variable region contains at least one of the LCDR1, LCDR2 and LCDR3 sequences, wherein:

[0043] The amino acid sequence of the LCDR1 has the amino acid sequence shown in SEQ ID No. 4: SSSVSSSF.

[0044] The amino acid sequence of the LCDR2 has the amino acid sequence shown in SEQ ID No. 5: RTS.

[0045] The amino acid sequence of the LCDR3 has the amino acid sequence shown in SEQ ID No. 6: QQWSGYPWT.

[0046] Or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6, or an amino acid sequence having more than 90% identity with SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6.

[0047] The anti-idiotypic antibody is a murine antibody, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 7:

[0048] QVQLQQSGAELARPGASVKLSCKASDYTFTNYWMQWVKQRPGQGLEW IGAIYLGDGDTRYTQKFKGKATLTADKSSSTVYMELNSLTSEDSAIYYCARDG WYFDVWGAGTTVTVSS. That is, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (116aa).

[0049] The amino acid sequence of the light chain variable region is as shown in SEQ ID No.8: ENVLTQSPAIMAASLGQKVTMTCSASSSVSSSFLHWYQQKSGASPKPLIHRTSK LVSGVPARFSGSGSGTSYSLTISSVEAEDDATYYCQQWSGYPWTFGGGTKLEIK. That is, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (108aa).

[0050] The binding portion of the above anti-idiotype antibody is selected from: F(ab’)2 fragment

[0051] The above anti-D antibody is a primate anti-D antibody; preferably, the primate anti-D antibody is selected from human anti-D antibody or monkey anti-D antibody.

[0052] The anti-idiotype antibody is a murine antibody. The constant region of the heavy chain of the anti-idiotype antibody is the heavy chain constant region of murine IgM, IgG1, IgG2a, IgG2b or IgG3, and the constant region of κ or λ type light chain.

[0053] As a specific embodiment, the present invention provides a murine anti-idiotype antibody 491# for detecting and neutralizing anti-D antibody. 491# is an artificial number.

[0054] In the present invention, hybridoma cells are screened, and the antibody secreted by them can bind to anti-D antibody protein. In the present invention, the type and subtype of the anti-idiotype antibody can be determined by using an antigen specific to a specific antibody type and subtype, and the mimic RhD antigen isotype can be determined by using an ELISA assay.

[0055] An anti-idiotype antibody for detecting and neutralizing anti-D antibody according to the present invention further comprises a murine antibody constant region. The murine antibody constant region includes the heavy chain constant region of murine IgM, IgG1, IgG2a, IgG2b or IgG3, and the constant region of κ or λ type light chain. An anti-idiotype antibody for detecting and neutralizing anti-D antibody is a primate anti-D antibody, and the primate anti-D antibody is selected from human anti-D antibody or monkey anti-D antibody.

[0056] The present invention also discloses a method for preparing an anti-idiotype antibody for detecting and neutralizing anti-D antibody, comprising the following steps: culturing host cells under expression conditions to express an anti-idiotypic monoclonal antibody of anti-D antibody; separating and purifying the expressed anti-idiotypic monoclonal antibody of anti-D antibody. Based on the affinity purification of the anti-idiotype antibody of anti-D antibody, such as non-denaturing gel purification, HPLC or RP-HPLC, purification on a protein A column, the anti-idiotypic monoclonal antibody of anti-D antibody is purified into a substantially homogeneous substance, for example, a single band on SDS-PAGE electrophoresis.

[0057] Example 1

[0058] Purification of anti-D antibody: The crude product of the anti-D antibody eluate from red blood cells was purified by Protein A affinity chromatography, and the anti-D antibody was obtained by elution. The harvested antibody molecules were identified by SDS-PAGE electrophoresis, and the molecular weight was consistent with the expected value, as shown in Figure 1 A. The anti-D antibody was ultrafiltered and replaced with solvent into a 50 mM citric acid buffer at pH 3.0, and digested with pepsin at 37 °C for 2 h. The Fc fragment and intact undigested protein were removed by Mabsure LX purification. The flow-through was collected to remove pepsin and other antibody fragments through a Capto L purification column, and the F(ab')2 protein was obtained by elution. The harvested protein was identified by SDS-PAGE electrophoresis. As can be seen from Figure 1 B, the reduced molecular weight of the target protein F(ab')2 segment was 25 kD, and the non-reduced molecular weight was about 100 kD, which was consistent with the expected value and could be used for subsequent immunization experiments.

[0059] Example 2

[0060] Preparation of anti-idiotype antibody hybridoma:

[0061] I. Female Balb / c mice (Beijing Huafukang Biotechnology Co., Ltd.) at 6 - 8 weeks old were subcutaneously injected with anti-D-F(ab')2 protein emulsified with complete Freund's adjuvant, 50 μg / dose / animal, and repeated three times. After the mice were subcutaneously immunized three times, 150 μl of blood was collected from the orbital sinus, and the serum was obtained after centrifugation. The enzyme-linked immunosorbent assay (ELISA) plate was coated with anti-D-F(ab')2 protein at a concentration of 1 μg / ml, and the antibody titer of the mouse serum was detected by ELISA. The mouse serum was diluted starting from 1:4000, and five concentrations were diluted 1:3. PBS was used as a negative control, and the optical density was detected at a wavelength of 450 nm by an enzyme-labeled instrument. As can be seen from Figure 2 this, the immune effects of the three mice were comparable, and the abundances of the serum antibody titers were 1#, 2#, and 3# in descending order.

[0062] II. Before fusion, the mice were intraperitoneally injected with 50 μg of anti-D-F(ab')2 protein for three consecutive days. One day before fusion, peritoneal macrophages from ordinary Kunming mice (Beijing Huafukang Biotechnology Co., Ltd.) were used as the feeder layer and inoculated into 96-well plates. The spleen of the immunized mice was fused with the non-secreting myeloma cell line SP2 / 0, and the hybridoma cells were added to the 96-well plates with the feeder layer paved, and HAT selection was performed on the fused hybridoma cells.

[0063] III. After the hybridoma cells grew in HAT medium for 12 days, 50 μl of the hybridoma supernatant was taken, and the antibody titer in the supernatant was detected by ELISA, and positive, negative, and blank controls were set. All positive clones were pooled, and after changing the HAT culture medium for two days, ELISA screening was performed again, and the clones that were positive both times were taken.

[0064] Example 3

[0065] Anti-idiotype antibody screening: Screening was performed by measuring OD620 with an ELISA reader. The specific method was as follows: Take a 96-well ELISA plate, add 75 μl of hybridoma supernatant and 5 μl of anti-D antibody, incubate at room temperature for 10 min, then add 20 μl of type O red blood cells to keep the reaction system at 100 μl. Set up a positive control for agglutination without adding hybridoma supernatant and a negative control for the secondary antibody. After mixing, incubate in a 37 °C incubator for 0.5 h. Wash the red blood cells with physiological saline, centrifuge at 200 g for 5 min, and repeat the washing twice. Add 100 μl of anti-human IgG secondary antibody, centrifuge at 300 g for 5 min, gently shake, visually observe the agglutination state of the red blood cells, and measure the optical density value with an ELISA reader at OD620. The results are as Figure 3 shown. The secondary antibody could not cause agglutination of type O red blood cells, and the red blood cells were evenly distributed in the wells of the plate; in the positive agglutination wells, the red blood cells aggregated on the side walls of the wells, resulting in increased light transmittance in this well. The target antibody was the clone with low light transmittance and high OD620 value. The wells marked in red were the candidate target clones. After multiple screenings of the hybridoma cells, candidate clones were obtained.

[0066] Using the limiting dilution method, the candidate hybridoma cells were subcloned. The 96-well cell culture plate was pre-coated with feeder cells to provide nutritional support for the growth of monoclonal hybridoma cells. After centrifugation of the candidate hybridoma cells in the exponential growth phase, the cell culture medium was adjusted in concentration so that the number of cells per well was 0.8 cells / well. After the hybridoma cells formed clones, positive clones were screened by the ELISA method, and monoclonal hybridoma cells were obtained.

[0067] Example 4

[0068] Determination of the affinity between anti-idiotype antibody and antigen:

[0069] Preparation of anti-idiotype antibody: After the hybridoma cells were amplified and cultured, the cell concentration was adjusted to 2×10^6 / ml with PBS and injected into the abdominal cavities of male 6-8-week-old Balb / c mice, 1 ml per mouse. Ascites was collected on the 10th day. After centrifugation of the ascites, the supernatant was filtered through a 0.45 μM filter to remove impurities, and the antibody in the supernatant was purified by Protein A. SDS-PAGE identification of the purified antibody showed that the molecular weights of the heavy and light chains of the antibody were correct, and it was Figure 4 known that the antibody purity was greater than 95%.

[0070] The Fortebio method was used to quantitatively detect the affinity between anti-idiotypic antibody and immunogen. An AMC probe was used to bind to the Fc segment of the idiotype antibody to detect its affinity and dissociation with the immunogen anti-D-F(ab')2. The ForteBio Octetred 96e molecular interaction analysis system is an advanced non-labeled and real-time monitoring technology mainly used for the comprehensive quantitative analysis of biomolecular interactions. An AMC probe was used to bind to the Fc segment of the idiotype antibody to detect its affinity and dissociation with the immunogen anti-D-F(ab')2. It is known that Figure 5 the anti-idiotypic antibody can bind to the anti-D-F(ab')2 molecule, has a strong binding force with the anti-D antibody, hardly dissociates, the dissociation curve is flat, and the calculated affinity (KD) is less than 1.0E-12 by fitting calculation.

[0071] Example 5

[0072] Experiment on the neutralization effect of anti-idiotypic antibody on red blood cell agglutination:

[0073] Two sources of anti-D antibodies were selected: IgG-D and IgM / G-D. IgG-D was selected from Shanghai Blood Biopharmaceutical Co., Ltd., National Medical Device Registration Approval: 20223401104, and IgM / G-D was selected from Millipore Corporation, Clones: TH-28 / MS-26. Take a 96-well plate, add anti-idiotypic antibody and anti-D antibody, with the usage amount of 5 μl or 10 μl, the final concentration of anti-idiotypic antibody is 500 μg / ml, incubate at room temperature for 10 min, then add 20 μl of type O red blood cells, keep the reaction system at 100 μl, set up a red blood cell blank control and a secondary antibody negative control, mix well and incubate in a 37 °C incubator for 0.5 h, wash the red blood cells with 200 μl of normal saline and centrifuge at 200 g for 5 min, repeat the washing 2 times, add 100 μl of anti-IgG secondary antibody, centrifuge at 300 g for 5 min and then shake. In order to more intuitively present the red blood cell agglutination state, use the automatic scanning and photographing function of the Elisapot instrument to display the red blood cell agglutination state. It is known that Figure 6 compared with the secondary antibodies 5C and 5D, both 5 ml or 10 ml of anti-D antibody can cause significant red blood cell agglutination effects 5A, 5B and 5G, 5H; among them: "5" represents the fifth column in the figure; A, B, C, D, G and H represent the row numbers in sequence. Taking 5A as an example, it represents the picture at the intersection of the "5" column and the A row.

[0074] It is known from Figure 6 the observations of 1C, 2C, 3C, 4C in that the anti-idiotypic antibody has a neutralizing effect on Shanghai IgG-D; according to the results of 6C and 7C, it has no antagonistic effect on IgM / G-D of Millipore Corporation, indicating that the anti-idiotypic antibody prepared by the present invention has selective neutralizing activity.

[0075] Example 6

[0076] The hybridoma antibody subtype was determined using a mouse monoclonal antibody subtype identification kit (Proteintech, Cat. PK20002). Take 50 μl of the hybridoma supernatant and dilute it 1:50 with 1×PBST. Add the test sample into the sample wells of the strip, 50 μL / well. Then add 1×goat anti-mouse IgM+IgG-HRP into the sample wells, 50 μL / well. Gently mix on a mixer and incubate at room temperature for 1 h. Discard the liquid in the wells, wash the plate three times with 1×PBST, and pat dry on absorbent paper. Add the freshly prepared chromogenic solution into the wells, 100 uL / well, and develop color at room temperature in the dark for 10 - 20 min. Add the stop solution into each well, 100 μL / well. Read the OD450 with an ELISA reader, and the well with the darkest color or the highest OD value corresponds to the corresponding subtype. The results are shown in Table 1.

[0077] Table 1. Identification of the subtype of murine anti-idiotypic antibody against D

[0078]

[0079] The experimental steps for sequencing the variable region gene of the anti-idiotypic antibody are briefly described as follows: Total RNA was extracted from hybridoma cells, and the first strand of cDNA was synthesized using reverse transcriptase (TaKaRa, Cat#2690A). According to the antibody subtype of the hybridoma cells, corresponding primers were selected for PCR amplification of the light / heavy chain. The specific bands amplified by PCR were recovered by gel cutting and cloned into the TA / Blunt-Zero (Vazyme Biotech Co., Ltd., Cat No.C601-01) vector. At least five colonies of the antibody were sequenced, and the sequencing results were analyzed by IgBLAST to determine the CDR region sequence of the antibody.

[0080] Example 7

[0081] Recombinant expression and identification of the anti-idiotypic antibody of the anti-D antibody:

[0082] 7.1 Construction of a murine recombinant antibody vector:

[0083] The obtained heavy chain and light chain nucleic acid variable region sequences were synthesized by Jiangsu Saisuofei Biotechnology Co., Ltd. Using homologous recombination technology, the sequences were respectively cloned into the murine IgG1 backbone eukaryotic expression heavy chain vector (Beijing Immune Ark Pharmaceutical Technology Co., Ltd., Cat No. pQKXM14) to obtain the corresponding heavy chain expression vector, and the light chain expression vector (Beijing Immune Ark Pharmaceutical Technology Co., Ltd., Cat No. pQKXM15) to obtain two anti-idiotype antibody light chain expression vectors. The HEK293 cells (ATCC, Cat No. CRL-1573) were cultured under the conditions of OPM-293CD05 serum-free medium (OPM, Cat No. 81075-001), 36.5 °C, 7.5% CO2, and suspended culture at 120 rpm. During transfection, the recombinant light paired heavy chain plasmid was mixed in 10 mL of OPM-293CD05 medium at a weight ratio of 1:1 (total DNA amount was 100 μg), and then 100 μL of PEI (concentration 3 mg / mL) was added, and quickly vortexed and mixed evenly, and incubated at room temperature for 15 minutes. Then this mixture was added to the above cell culture. After culturing the cells for 7 days, the antibody in the supernatant was harvested to obtain the cell culture.

[0084] 7.2 Purification of the recombinant antibody:

[0085] The harvested cell culture was centrifuged at 3000×g for 20 min, the supernatant was collected and filtered through a 0.45 μm filter. The antibody was purified using a 5 mL Protein A affinity chromatography column (GE), eluted with 50 mM citric acid (pH 3.0) buffer at a flow rate of 5 mL / min, and the complete elution peak was collected. At the same time, the pH of the collected eluate was adjusted to about 7.0 with 1 M Tris HCl (pH 9.0) buffer. The obtained protein was detected by SDS-PAGE and Coomassie Brilliant Blue staining. As shown in A in Figure 7 it, the reduced molecular weights of the target proteins were 50 KD and 25 kD, indicating that the target proteins were of IgG type.

[0086] 7.3 ELISA identification of the recombinant anti-idiotype antibody:

[0087] ELISA was used to detect the binding of the anti-idiotype antibody to the immunogen. The ELISA plate was coated with anti-D-F(ab')2 protein at a concentration of 1 μg / ml, and the murine recombinant antibody concentration was 1 μg / ml. 100 μl of each was added to the ELISA plate, and PBS solution was used as a blank control. The secondary antibody was HRP-labeled goat anti-mouse IgG (Suzhou Botelong Immunotechnology Co., Ltd., Cat No. BF03001), and the optical density was detected at a wavelength of 450 nm by an enzyme-labeled instrument. The results are shown in Table 2. The prepared recombinant anti-idiotype antibody bound to the antigen, indicating that the variable region sequence was correct.

[0088] Table 2

[0089]

[0090]

[0091] 7.4 Hemagglutination identification of recombinant anti-idiotype antibody on red blood cells:

[0092] Take a 96-well plate, add 50 μg of recombinant anti-idiotype antibody and 5 μl of IgG-D antibody respectively, incubate at room temperature for 10 min, then add 20 μl of type O red blood cells, and keep the reaction system at 100 μl. Set up a red blood cell blank control and a secondary antibody negative control. After mixing, incubate in a 37 °C incubator for 0.5 h. Wash the red blood cells with 200 μl of physiological saline and centrifuge at 200 g for 5 min, repeat the washing 2 times. Add 100 μl of anti-IgG secondary antibody, centrifuge at 300 g for 5 min, then gently shake, observe and record the red blood cell agglutination status under a microscope. As can be seen from Figure 7 B in the figure, there was no red blood cell agglutination in the red blood cell blank control and the secondary antibody negative control groups, and 5 μl of anti-D antibody could cause obvious red blood cell agglutination; after premixing 50 μg of anti-idiotype antibody with anti-D antibody, compared with the corresponding anti-D antibody group, it can be known that the recombinant anti-idiotype antibody has the effect of neutralizing the hemagglutination of red blood cells mediated by anti-D antibody.

Claims

1. An anti-idiotypic antibody that neutralizes anti-D antibodies, characterized in that comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 sequences, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 sequences, wherein: The amino acid sequence of the HCDR1 is shown in SEQ ID No. 1; The amino acid sequence of the HCDR2 is shown in SEQ ID No. 2; The amino acid sequence of the HCDR3 is shown in SEQ ID No. 3; The amino acid sequence of the LCDR1 is shown in SEQ ID No. 4; The amino acid sequence of the LCDR2 is RTS; The amino acid sequence of the LCDR3 is shown in SEQ ID No.

6.

2. The anti-idiotypic antibody for neutralizing anti-D antibodies according to claim 1, wherein The amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 7; the amino acid sequence of the light chain variable region is shown in SEQ ID No.

8.

3. The anti-idiotypic antibody for neutralizing anti-D antibodies according to claim 2, wherein The anti-idiotypic antibody is a mouse antibody.

4. The anti-idiotypic antibody for neutralizing anti-D antibodies according to claim 3, wherein The constant region of the heavy chain of the anti-idiotypic antibody is the heavy chain constant region of mouse IgM, IgG1, IgG2a, IgG2b or IgG3, and the constant region of the light chain of the anti-idiotypic antibody is of κ or λ type.

5. The anti-idiotypic antibody for neutralizing anti-D antibodies according to claim 4, wherein The antigen binding portion of the anti-idiotypic antibody is selected from F(ab')2 fragments.

6. Use of an anti-idiotypic antibody for neutralizing an anti-D antibody according to any one of claims 1 to 5, characterized in that: The anti-idiotypic antibody is used to prepare a reagent for detecting the content of anti-D antibodies in serum.

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